Selective and Differential Agars for Gram-Negative Rods

    In our laboratory we examine the characteristics of several different varieties of gram-negative microorganisms.  One of the groups we focus on are those belonging to the family Enterobacteriaceae.  This group includes many species that may be found in the gastrointestinal tracts of animals.  To separate those that are gastrointestinal flora from other groups and from gram-positive microorganisms, it is possible to take advantage of ecological differences in the chemical makeup of the environment in which they live.  Bile salts, found in the gastrointestinal tract of mammals, inhibit the growth of many bacterial species, especially most gram-positives.  The addition of bile salts to microbiological media makes these media selective for gram-negative rods and only those few gram-positive species that live in similar environments.

    Many of the gram-negative flora of the gastrointestinal tract of mammals belong to a group called coliforms, since they are found in large numbers in the colon and resemble the characteristic species Escherichia coli.  Coliforms can be differentiated from other G- rods based on their ability to ferment the milk sugar lactose.  Non-coliform rods such as Salmonella typhimurium, a pathogen normally found in the gastrointestinal tract of reptiles and birds, and Proteus mirabilis, an opportunistic pathogen of the urinary tract, lack the enzymes necessary to metabolize this sugar.  By adding lactose and one or more pH indicators to a selective medium for G- rods, we can easily differentiate between the two groups.

MacConkey's Agar

Maconkey's agar is a medium that contains bile salts and crystal violet as its selective agents.  These inhibit the growth of most gram-positives.  It also contains lactose and a neutral red indicator that turns pink in the presence of the metabolic by-products of lactose fermentation.  In this image, the two lactose-fermenting coliforms E. coli and E. aerogenes have produced the characteristic pink color, while S. typhimurium and P. mirabilis, the two non-coliforms have not.

Hektoen Agar

Hektoen agar is also used to select for an differentiate between groups of gram-negative rods.  Hektoen agar contains bile salts, lactose, acid fuschin and brom thymol blue indicators.  It also contains sodium thiosulfate and ferric ammonium citrate, used to determine hydrogen sulfide (H2S) production.  Coliform rods will produce colonies that range in color from yellow to orange-yellow, while non-coliform colonies remain the green color of the agar itself.  Organisms such as S. typhimurium have the ability to utilize sulfate as a final electron and hydrogen acceptor during anaerobic respiration, producing H2S as a by-product.  This undergoes an exchange reaction with ferric ammonium citrate, producing ferric sulfate (FeS), a black colored precipitate.  Given time, the green colonies of S. typhimurium will develop a black center, thus are sometimes referred to as "fish eye" colonies.